Short Communication

J Mol Microbiol Biotechnol 2010;18:156–161 Published online: April 13, 2010 DOI: 10.1159/000308518

Rapid Detection and Identification of casei ATCC 393 by Multiplex PCR

Athanasios Karapetsas Eleftherios Vavoulidis Alex Galanis Raphael Sandaltzopoulos Yiannis Kourkoutas

Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis , Greece

Key Words and the World Health Organization (WHO) of the Unit- ؒ ؒ Multiplex PCR ؒ ed Nations defined as ‘live microorganisms Probiotics which when administered in adequate amounts confer a health benefit on the host’ [FAO, 2002]. Maintenance of the intestinal microbial homeostasis, prevention of patho- Abstract genic infections, stabilization of the gastrointestinal bar- Many functional foods containing probiotic strains have rier function and production of anti-carcinogenic and been developed recently. Lactobacillus casei ATCC 393 is one anti-mutagenic compounds [Choi et al., 2006; Boirivant of the most frequently used cultures in probiotic products. and Strober, 2007; Saulnier et al., 2008] are included The present study aimed to develop a method for the detec- among the beneficial effects of probiotic-based foods, tion and identification of L. casei ATCC 393 based on genetic mainly yogurt and other dairy products. Thus, a probi- polymorphisms of the hsp60 gene. A multiplex polymerase otic-rich diet is linked with the prevention and potential chain reaction (PCR) assay was designed, utilizing two novel treatment of several severe digestive disorders, such as strain-specific primer sets that enable identification of L. ca- inflammatory bowel disease [Boirivant and Strober, sei ATCC 393. The accuracy of our method was further con- 2007], colorectal cancer [Fotiadis et al., 2008] and ob- firmed by successful identification of our strain in probiotic structive jaundice [unpubl. data]. cheese. The method described is an easy to use, rapid, inex- Lactobacillus and Bifidobacterium species are the most pensive and accurate tool that may be readily applied to commonly used probiotic microorganisms. Among these food, fecal and intestinal samples. (LAB), the Lactobacillus casei ATCC Copyright © 2010 S. Karger AG, Basel 393 strain has been extensively incorporated into food products to confer probiotic properties [Kourkoutas et al., 2005, 2006; Li et al., 2009]. Although the taxonomic In recent years, the development and production of position of L. casei ATCC 393 (called L. zeae ATCC 393 novel foods containing probiotic microorganisms have in previous literature) was a subject of controversy in the attracted tremendous interest due to their healthful prop- past, the Judicial Commission recently affirmed that typ- erties. The Food and Agriculture Organization (FAO) ification of L. casei is based on ATCC 393 [Judicial Com-

© 2010 S. Karger AG, Basel Dr. Y. Kourkoutas 1464–1801/10/0183–0156$26.00/0 Dr. R. Sandaltzopoulos Fax +41 61 306 12 34 Department of Molecular Biology and Genetics, Democritus University of Thrace E-Mail [email protected] Accessible online at: GR–68100 Alexandroupolis (Greece) www.karger.com www.karger.com/mmb E-Mail ikourkou @ mbg.duth.gr; rmsandal @mbg.duth.gr L. zeae GGCGACCAAGGCAGCG CTTCGGTTTCATCTTCC CTGATTGCGGACGCC L. paracasei AGCAACTAAGGCTGCC CGTCCGTTTCTTCCTCA CTGATTGCCGACGCC L. ingluviei GGCTACGGCCGCAGCC CTTCGATCTCTGCTGCG TTGATTGCGGACGCA Fig. 1. The hsp60 gene sequence contain- L. kefiri GGCTACCGAGACTGCT CTTCTGTTTCATCAGCT TTAATTGCTGATGCA AGCTACTGCAGCCGTA CTTCAGTTTCATCATCT TTAATCGCTGACGCT ing the diagnostic nucleotides (in bold) L. acetotolerans targeted by the novel primers designed in L. casei ATCC 393 5’… GGCGACCAAGGCAGCG78 CTTCGGTTTCATCTTCC156 CTGATTGCGGACGCT 189 …3’ this study.

mission of the International Committee on Systematics of Bacteria, 2008]. Unfortunately, experimental analysis and quality con- trol are hampered by the dire lack of a rapid, reliable and sensitive method for the detection of the above strain, in DSM 20749 DSM 5622 order to confirm and establish the probiotic character of DSM 15946 ATCC 393 ATCC DSM 20587 a product. Identification and characterization of LAB in DSM 20178 probiotic products are based on microbiological and mo- lecular techniques. However, conventional microbiologi- DNA ladder L. casei L. kefiri L. acetotolerans L. ingluviei L. zeae L. paracasei cal and biochemical tests for phenotypical characteriza- tion are considered inadequate, as they have limitations in discriminating large numbers of isolates with similar 500 bp physiological characteristics [Mohania et al., 2008]. On 300 bp the other hand, several -based methods have re- cently been developed. These methods are more accurate, 100 bp sensitive, time-efficient and capable of identifying a wide spectrum of LAB in a single reaction [Mohania et al., 123456 2008]. They are either directed at unique genes specific for a microbial species or at unique sequences of ubiqui- tous genes. Likewise, a number of studies focusing on the Fig. 2. Agarose gel electrophoresis of PCR products from multi- plex PCR assay. identification of Lactobacillus species developed poly- merase chain reaction (PCR)-based molecular assays em- ploying primers that target the variable regions of the 16S rRNA, the 23S rRNA, the 16S-23S rRNA intergenic spac- ferent Lactobacillus species and the wealth of sequence er region (ISR) or other universal genes [Nour et al., 1998; data available in GenBank. Ward et al., 1999; Fasoli et al., 2002; Teanpaisan et al., The hsp60 gene sequences of all 56 Lactobacillus 2006]. In parallel, a multiplex PCR assay system has been strains available in GenBank, including the L. casei developed that allows the simultaneous amplification of ATCC 393 strain, were obtained by the National Center two or more loci in the same reaction, thus increasing for Biotechnology Information (NCBI) (table 1). The se- sensitivity and accuracy of discrimination [Song et al., quences were aligned against each other using the DNA 2000; Settanni et al., 2005; Sul et al., 2007]. sequence alignment tool, ClustalW2 (European Bioinfor- The aim of the present study was to develop a multi- matics Institute – EMBL). Extensive analysis allowed the plex PCR assay to detect and unequivocally identify the identification of polymorphic sites that are unique for the L. casei ATCC 393 strain in a single reaction. Our meth- L. casei ATCC 393 strain. The development of a multiplex odology was based on the hsp60 gene sequence polymor- PCR assay that enables straightforward detection of the phisms. Polymorphic sites within the hsp60 gene have strain of interest was based on the interrogation of com- been used successfully as a molecular marker [Goh et al., binations of three nucleotide sites, at positions 78, 156 1996; Blaiotta et al., 2008]. The main advantage of this and 189 (position +1 corresponds to the first nucleotide gene over other ubiquitous genes is the higher resolution of the cDNA). These sites were selected because they have that may be achieved in species discrimination, due to the the greatest discriminatory value. As shown in table 1, L. great variability of the hsp60 nucleotide sequences of dif- casei ATCC 393 was the only strain with a G-C-T base

L. casei ATCC 393 Detection by J Mol Microbiol Biotechnol 2010;18:156–161 157 Multiplex PCR Table 1. List of Lactobacillus strains included in this study

Reference strains Collection No. Accession No. Bases at diagnostic sites (78, 156, 189)

Lactobacillus acetotolerans DSM 20749 AF429666 A-T-T Lactobacillus acidipiscis DSM 15836 AJ621719 T-A-T Lactobacillus acidophilus DSM 20079 AY424311 T-A-T Lactobacillus alimentarius DSM 20249 AY424318 T-A-T Lactobacillus amylophilus DSM 20533 AY424319 G-A-C Lactobacillus amylovorus DSM 20531 AY424312 C-A-T Lactobacillus bifermentans DSM 20003 AY424320 T-T-C Lactobacillus brevis DSM 20054 AY424329 T-A-C Lactobacillus buchneri DSM 20057 AY571676 T-C-A Lactobacillus casei ATCC 393 AY424336 G-C-T Lactobacillus casei ATCC 11578 n.a. n.a. Lactobacillus casei DN114001 n.a. n.a. Lactobacillus casei BB-12 n.a. n.a. Lactobacillus casei CCC B1241 AF429642 C-A-C Lactobacillus casei DSA-FB2 AY424335 C-A-C Lactobacillus casei ATCC 4913 AF429654 T-A-C Lactobacillus casei CCC B1205 AF429631 C-A-C Lactobacillus casei DSA 145 AY424333 C-A-C Lactobacillus casei ATCC 334 AY424332 C-A-C Lactobacillus casei CCC 95G2L AF429701 C-A-C Lactobacillus casei DSA 15 AY424334 C-A-C Lactobacillus casei I18C AF429649 C-A-C Lactobacillus casei CCC B9657 AF429644 C-A-C Lactobacillus casei I03 AF429647 C-A-C Lactobacillus coryniformis DSM 20001 AY424321 C-T-T Lactobacillus crispatus DSM 20584 AY424313 T-T-T Lactobacillus curvatus DSA 32Y AY424348 T-T-A Lactobacillus cypricasei DSM 15353 AJ621720 T-A-T Lactobacillus delbrueckii subsp. lactis DSM 20072 AY424322 C-T-C Lactobacillus durianis DSM 15802 AJ621721 T-T-T Lactobacillus farciminis DSM 20184 AY424323 A-T-C Lactobacillus fermentum DSA FB5 AY424325 C-T-C Lactobacillus fructivorans DSM 20203 AF429681 C-C-A Lactobacillus gallinarum DSM 10532 AY571675 T-T-T Lactobacillus gasseri DSM 20243 AY424314 T-T-A Lactobacillus helveticus DSM 20075 AY424315 T-T-T Lactobacillus hilgardii DSM 20571 AF429699 T-T-A Lactobacillus homohiochii ATCC 15434 AF429685 T-T-T Lactobacillus ingluviei DSM 15946 AB267877 C-G-A Lactobacillus jensenii DSM 20557 AF429687 T-T-T Lactobacillus johnsonii DSM 20553 AY424317 T-T-A Lactobacillus kefiranofaciens DSM 5016 AF429691 C-A-T Lactobacillus kefiri DSM 20587 AF429688 T-T-A Lactobacillus kimchii DSM 13961 AY571674 T-T-C Lactobacillus mali DSM 20444 AF429692 A-C-A Lactobacillus parabuchneri DSM 5708 AF429638 A-C-A Lactobacillus paracasei DSM 5622 AY424340 C-A-C Lactobacillus paracasei DSM 20006 n.a. n.a. Lactobacillus paracasei DSM 20207 n.a. n.a. Lactobacillus paracasei DSM 46331 n.a. n.a. Lactobacillus paracasei DSM 20312 n.a. n.a. Lactobacillus paracasei DSM 20008 AF429694 C-A-C Lactobacillus paraplantarum DSM 10667 AY424357 G-A-C Lactobacillus pentosus DSM 20314 AY424353 G-A-C Lactobacillus plantarum DSM 13273 AY571677 G-A-C

158 J Mol Microbiol Biotechnol 2010;18:156–161 Karapetsas/Vavoulidis/Galanis/ Sandaltzopoulos/Kourkoutas Table 1 (continued)

Reference strains Collection No. Accession No. Bases at diagnostic sites (78, 156, 189)

Lactobacillus reuteri DSM 20016 AY424326 T-T-A DSM 20021 AY424337 C-T-A Lactobacillus sakei LTH 673 AY424352 T-T-T Lactobacillus salivarius DSM 20554 AY424328 A-T-C Lactobacillus sanfranciscensis DSM 20451 AY700220 C-C-C Lactobacillus thermotolerans DSM 14792 AJ621723 C-A-A Lactobacillus vaccinostercus DSM 20634 AJ621724 T-T-C Lactobacillus zeae DSM 20178 AY571673 G-C-C

The collection number relates to the Lactobacillus strain registered to the microbial banks. The accession number applies to the hsp60 gene of each strain. The strains used in the present study are indicated in bold. n.a. = Accession number and sequence data not available in NCBI.

Table 2. Oligonucleotide primers used in this study strains may carry the same base as L. casei at one or at two but not at all three of these diagnostic positions. ؅ ؅ Primer Primer sequence (5 K3 ) Length Optimal Therefore, multiplex PCR allows the exclusive identifica- bp amount pmol tion of L. casei with enhanced accuracy and reliability. According to the hsp60 gene sequences of all strains ex- p78F GGCGACCAAGGCAGCG 16 10 amined, these primers do not generate any PCR fragment p156F CTTCGGTTTCATCTTCC 17 50 unless the L. casei ATCC 393 strain genomic DNA is used p189R GGCCAACTTTTTCCATA 17 50 as template. In addition, a set of primers (LacF and LacR) LacF AGCAGTAGGGAATCTTCCA 19 10 LacR ATTYCACCGCTACACATG 18 10 that recognize the 16S rRNA gene of all Lactobacillus strains was included in the present study as a positive control marker [Walter et al., 2001]. The specificity of the primers and the expected size of PCR products are pre- Table 3. Specificity of primers designed for multiplex PCR on Lac- sented in table 3. The annealing positions of these prim- tobacillus strains ers were designed so that they generate fragments which can be easily resolved in a standard agarose gel electro- Reference strain Specificity of primer pairs phoresis. p78F/ p156F/ LacF/ In order to rigorously test the reliability of our PCR- p189R p189R LacR based methodology, we employed 5 strains selected in (144 bp) (67 bp) (340 bp) such a way that their sequences represent every possible L. casei ATCC 393 +++nucleotide composition at the diagnostic positions (fig. 1 ; L. acetotolerans DSM 20749 ––+table 1). The strains were provided by the Deutsche L. ingluviei DSM 15946 ––+Sammlung von Mikroorganismen und Zellkulturen L. paracasei DSM 5622 ––+(DSMZ) microbial bank (Braunschweig, Germany) and L. zeae DSM 20178 ––+grown on MRS broth (Merck, Darmstadt, Germany). Ge- nomic DNA from liquid cell cultures was extracted using The expected size of PCR products is indicated. a DNeasy Tissue Kit (Qiagen, Hilden, Germany) accord- ing to the manufacturer’s protocol. The amount of ex- tracted DNA was determined by absorbance at 260 nm composition at these three positions respectively. Thus, using a UV spectrophotometer (Eppendorf). All reac- we designed two sets of primers (p78F-p189R and p156F- tions were carried out in a total volume of 50 ␮l, contain- p189R) that are complementary to the sequence of the ing 5 units Taq DNA polymerase (HyTest Ltd.), 400 ␮M hsp60 gene of the strain of interest ( table 2 ). All primers each dNTPs (Promega), 1.5 m M MgCl2 (HyTest Ltd.) and end at diagnostic nucleotide positions. Some other Lacto- 100 ng template DNA. Optimized amounts of primers

L. casei ATCC 393 Detection by J Mol Microbiol Biotechnol 2010;18:156–161 159 Multiplex PCR DSM 20006 DSM 20207 DSM 46331 DSM 20312 DSM 20008 DN114001 BB-12 ATCC 393 ATCC ATCC 11578 ATCC ATCC 393 ATCC DNA ladder L. casei product Commercial L. casei product Commercial L. casei DNA ladder L. casei L. paracasei L. paracasei L. casei L. paracasei L. paracasei L. paracasei

500 bp 500 bp

300 bp 300 bp

100 bp 100 bp

a 123 45 67 b 1 23

Fig. 3. Multiplex PCR assay of L. casei and L. paracasei strains ( a) and two commercial probiotic products con- taining L. casei DN114001 and L. casei BB-12 ( b ).

per 50-␮ l reaction are shown in table 2. Amplification ysis was performed on 6 additional L. casei and L. para- was carried out in a Thermal Cycler (Mastercycler Ep- casei strains ( L. casei ATCC 11578, L. paracasei DSM pendorf) under the following conditions: 94 ° C (2 min), 20006, L. paracasei DSM 20207, L. paracasei DSM 46331, followed by 25 cycles of 94 ° C (15 s), 51 ° C (15 s), 72 ° C (30 L. paracasei DSM 20312 and L. paracasei DSM 20008) in s), followed by a final extension step at 72 ° C (1 min). The liquid cultures (fig. 3a) and in 2 commercial probiotic PCR products were separated on 1.5% w/v agarose gels, products ( fig. 3 b). It is worth mentioning that for most of visualized under UV illumination and photographed the above strains no sequence data was available (table 1). with a digital camera (Gel Doc EQ System Biorad). We then tested whether our method could be used for the To optimize primer concentrations and cycling condi- detection of L. casei ATCC 393 in probiotic cheese pre- tions (extension and annealing time and temperature), pared as described previously [Kourkoutas et al., 2006]. single PCR reactions were initially performed on L. casei Cheese samples were blended with sterilized ¼ Ringer ATCC 393, whereas the 5 representative Lactobacillus solution and total DNA was extracted from the liquid cell strains were used as negative controls. Subsequently, op- suspension as described above. Then, a multiplex PCR timization of the multiplex PCR assays was performed assay was performed using our novel strain-specific (data not shown) [Henegariu et al., 1997]. Consistently, primers. As expected, three PCR products were generated the multiplex PCR assays generated three products (67, (data not shown), corresponding to the unique pattern of 144 and 340 bp) for L. casei ATCC 393 and only one (340 L. casei ATCC 393 (fig. 2), thus confirming the presence bp) for any other Lactobacillus strain tested ( fig. 2 ). The of our strain in the cheese. Similarly, the applicability of two shorter fragments (67 and 144 bp) originate from the our method for the detection of L. casei ATCC 393 in in- hsp60 gene sequence that is characteristic of the L. casei testinal and fecal samples has been tested successfully. Of ATCC 393, whereas the 340-bp fragment corresponds to note, the proposed method was able to detect the strain the 16S rRNA sequence common in all Lactobacillus of interest on the above samples even at levels ^ 10 1 cfu/g. strains and serves as a positive control marker [Walter et This application illustrates the advantage of our approach al., 2001]. over classical microbiological methods that require mul- Under all conditions tested, L. casei was readily detect- tiple dilutions and culture in order to detect LAB content able. No false-negative result was encountered. Impor- in mixed microbial populations. Our multiplex assay tantly, no false-positive results were obtained when anal- readily identifies the exact strain without any require-

160 J Mol Microbiol Biotechnol 2010;18:156–161 Karapetsas/Vavoulidis/Galanis/ Sandaltzopoulos/Kourkoutas ment for separation of colonies. Our work paved the way require sophisticated lab equipment. It can be immedi- for future attempts to develop quantitative multiplex PCR ately adopted for detection and identification of this pro- assays which may allow determination of the exact mi- biotic strain in food products, intestinal samples and fe- crobial load in food or biological samples. ces. Additionally, our methodology can be easily adapted In conclusion, the proposed multiplex PCR method for other probiotic LAB species and strains, thus provid- proved an efficient tool for accurate, convenient and re- ing a powerful tool of molecular discrimination that liable identification of the Lactobacillus casei ATCC 393 could be invaluable in food industry. strain. Our approach is rapid, inexpensive and does not

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L. casei ATCC 393 Detection by J Mol Microbiol Biotechnol 2010;18:156–161 161 Multiplex PCR